ABSTRACT: Since primeval times, the inflammatory process has been described in many different ways. Several anti-inflammatory therapies have been used in different biological models. However, in a recent "back to nature move", modern man is searching for natural products with medicinal properties, particularly those obtained from plants and bees. Propolis has been used in folk medicine for a very long time. The many compounds present in propolis require investigation. Physical-chemical analysis studies have not sufficiently established quality standards of propolis containing products. These standards should depend especially on their different pharmacological activities. There are few studies reporting on the in vitro anti-inflammatory activity of propolis containing products. It is necessary to evaluate the anti-inflammatory potential of commercial products containing propolis. KEYWORDS: propolis, anti-inflammatory activity, bees, Apis mellifera, natural products.

INTRODUCTION

More than two thousand years ago, the ancient Greeks used the term phlogosis and the Romans inflammation to designate the same phenomenon nowadays called inflammation. Since the first description of this phenomenon by Aulus Cornelius Celsus, the inflammation process has been described in many different ways. In the Middle Ages, inflammation was thought to be the heat accumulation originating in the heart, followed by blood flow, mucous, and bile; this was the humoral inflammation theory. This was then overtaken by the vascular theory in the 18th century.

The concept of inflammation has evolved since the discovery of cells in the 19th century. By this time, inflammation was seen to be preceded by cell and tissue injuries, and that vascular changes including leukocyte emigration were secondary events (12,85).

During the 1920s, the idea that the vascular system facilitated quick accumulation of great quantities of phagocytes and antibodies was reviewed. The first physical-chemical analysis of inflammation, cell stress and local tissue changes, promoted by an increasing concentration of oxidants and osmotic pressure, were also made at this time (21). Therefore, modern investigators have considered inflammation a primary event of the host defense system.

Inflammation can be basically defined as a change of the morphological equilibrium in a specific area of the tissue caused by different kinds of agents: physical, chemical, or biological (141). It can be represented by capillary dilatation with fluid accumulation (oedema) and by phagocyte emigration and accumulation (neutrophils, monocytes, macrophages), which also contribute to hyperalgesia generation and loss of tissue function (42). Other characteristics, such as erithema and fever, can also be observed during inflammatory events. The last feature occurs after cytokine release (IL-1, TNF-a ) by activated macrophages, leading to a vessel dilatation resulting from smooth muscular relaxation and followed by an increase in local blood flow (hypothermia) (118,127). The increased hematocrit leads to an erythrocyte aggregation, and leukocytes move from the central axial of the vessel to the periphery ("Roleux") (19).

Similarly, many intra- and extracellular phospholipases are activated from the cytoplasmic membrane phospholipids and activate other enzymes, such as cyclooxygenase (COX) and lipoxygenase (LOX), which act on arachidonic acid (AA) and eicosanoid metabolism (105,106,138). The fibrinolytic system, kinins, complement, vaseactive amines (histamine and serotonin), and nitric oxide may lead to inflammation when physiologically altered (117,138).

Neuropeptides of the skin nerves may interact with target-cells in the skin, releasing more skin neuropeptides, such as P substance, vase intestinal peptides (VIP), and peptides regulator of calcitonin gene, which modulates not only the function of inflammatory and immunocompetent cells but also endothelial and epithelial cells (70).

Endothelial-leukocyte cellular adhesion occurs in a sequence of events, and specific molecules are expressed in different stages. Selectins (E, P, and L), integrins (VLA-4 and LFA-1), and members of immunoglobulin super-families (ICAM-1 and ICAM-2) move leukocytes from the vascular lumen to the tissues (34,93,135). In response to several mediators, the vascular endothelium expresses specific glycoproteins on the cell surface, which intermediate blood leukocyte connection and extravasation, important for tissue repair (10,31,124). In general, the irritating phlogogen agent, and the frequency and magnitude of body response have allowed a classification of inflammation into immunogenic (self) and non-immunogenic (non-self) (119); the latter is subdivided in acute and chronic phases (127).

The acute-phase response involves serous, fibrinous, supurative or exudative events as well as micro-vascular and cell events; this response to phlogogen occurs within 72 hours. The chronic-phase response includes proliferative events and histological alterations, different from those in the acute phase, characterized by cell emigration and intensive mitosis. Formation of giant multinuclear cells takes place, and all these events are induced by phlogogen (119). In addition, inflammation may be physiological or pathological (39), depending mainly on histological aspects. Specific-immune events, such as hypersensitive reactions (types I, II, III, and IV) can also lead to inflammation (103,122,123).

In spite of the above classifications, inflammation comprises a great variety of reactions occurring in the body, for instance arthritis. Several etiological and metabolic pathways are involved in the inflammatory response (96). All distinct inflammation events, culminating in oedema and pain in bone articulations are generally denominated arthritis. Inflammation studies require different experimental models, so that different metabolic pathways may be elucidated. Inflammation is an important causative agent of human morbidity and mortality, such as systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), and multiple organ failure (MOF) (8). In this way, inflammation events permit molecule identification and allow the development of drugs capable of acting on a variety of related metabolic pathways.

The action of several corticosteroids on asthma, rhinitis, and dermatitis is well known (6,7,88). These drugs have been used in anti-inflammatory therapy in clinical and pre-clinical assays (35,55,86). Like other hormones, corticosteroids act on many different tissues and body systems. At physiological concentrations, they maintain normal blood pressure, heart function, respond to inflammatory prostaglandin (PG) action, and maintain blood volume, diminishing vascular endothelium permeability (48,102). However, these effects are accentuated at high pharmacological concentrations, leading to target-cell dysfunction (mast cells, macrophages, vascular smooth muscles, and mucous glands) (38,48).

Glucocorticoids are used due to their general anti-inflammatory activity, which ranges from clinical suppression of rheumatoid arthritis to palliative treatment of some allergic manifestations, such as bronchitis, asthma, and anaphylatic reactions (6,7,44). They also act upon cytokines involved in eosinophil, basophil, and lymphocyte recruitment (112).

The activity of these compounds depends on the presence of a hydroxyl group in carbon-11 (102). This group acts in a similar manner to the anti-inflammatory activity of macrocortins, rinocortins, or lipocortins (101). Simultaneous findings have been reported on calcium anexin family members and protein phospholipids of several intrinsic groups in the phosphorilation control (25,45,104).

Glucocorticoid anti-inflammatory effects with the LC-1 protein act on arachidonic acid (AA) metabolites, phospholipase A2 (PLA2) inactivation, and COX and LOX (88). These events are present in triggering of apoptotic processes in various cell types, protection against splenic artery occlusion, and reperfusion of peritoneal macrophages (22,37) and neutrophils. However, the exact biological functions are not yet totally clear (59).

Induction of anti-inflammatory mechanisms is also present in synovial arthritis, though the ability to regulate articulation inflammation is still unclear (139,140). The inhibitory action of glucocorticoids on endothelium-leukocyte interaction affects the expression of cellular adhesion molecules (CAM).

Glucocorticoids inhibit the expression of ELAM-1 and ICAM-1 in the endothelium and suppress the expression of LFA-1 in stimulated lymphocytes (28,34,100). Toxicity is high during long-term anti-inflammatory therapy, which limits its unrestricted use (44).

The effects of non-steroid anti-inflammatory drugs (NSAID) on the synthesis of inflammatory prostaglandins, especially the PGE2, are widely known (105,106). The molecular mechanisms of these inflammation compounds have only recently been elucidated (99,128).

The pharmacological aim of most NSAID is the COX-enzyme (PGHS or PGH2) (66,99). Two isoforms of these enzymes are described, COX-1 and COX-2, differing in tissue expression and distribution (137). The mechanism of all NSAID studied until now is based on their involvement with the hydrophobic region of both isoforms; the manner in which the molecule structure of the drug relates to this region may vary (138).

Many other classes of COX-2 selective inhibitors have been developed: 1) sulfonamides (including NS-398, flosulide and L-745, 3337); 2) tricyclic methylsulfone derivatives (including DuP-697, SC-58125,and SC-57666) (131). In relation to LOX inhibitors, the monomethylamine analogs, such as LY269415 and LY-221068, are not only antioxidants but also strong inhibitors of iron-dependent lipidic peroxidation (91).

All the NSAID tested in animal models and man prevent pain, oedema, and erithema leading to reduced inflammatory reactions (138). Administration of therapeutic doses in rats is effective in reducing degenerative diseases associated with adjuvant-induced arthritis. In humans, NSAID have been considered very effective in acute and chronic inflammation, such as arthritis, tendinitis, and pericarditis (137). COX-1 inhibition by NSAID may cause side effects, such as gastrointestinal and renal disorders (129,131,134).

After the advent of glycobiology, clinical studies with CAM have allowed the development of anti-inflammatory strategies (34). Inflammatory reactions as glomerulonephritis, ulcerative colitis, syndrome of breath stress, rheumatoid arthritis, autoimmunity, and atherosclerosis partly result from the pathological activation of endothelial-leukocyte adhesion (97).

Recent studies have shown that mimetic CAM may block inflammation induction, though the differences of adhesion are not clear (10). In these cases, the following components are used: quimerics-immunoglobulins-selectins-proteins, carbohydrate, and peptide carbohydrates anti-sense oligonucleotides, and low-molecular weight inhibitors (13,32).

On the other hand, inhibition of FR production has been stimulated by endogenous antioxidant synthesis and administration of some factors and co-factors for exogenous antioxidant formation (98).

Physiological production of FR is related to: 1) activation and migration of phagocytic cells; 2) COX activation during AA metabolism; 3) catecholamine oxidation; 4) uric acid formation through xanthine oxidase; and 5) microsomal enzyme P-450 action on mitochondrial internal membrane through cytochrome oxidase complex by oxygen reduction. Oxygen is transported by hemoglobins (67); however, excessive quantities of FR are closely related to several inflammatory events, resulting in the production of oxygen toxic reactive species (OTRS) (136).

Natural antioxidant mechanisms (enzymatic or non-enzymatic) have been stimulated. Small molecules and kelants of metallic ion (selenium, zinc, copper, manganese, vitamins A, C, and E, cysteine, and reduced glutathione, and some plasma compounds) participate in inflammatory events and act on ORTS (11,24). Similarly, in vitro inhibition of PMN adhesion by SOD and micro-vascular extravasation by SOD catalysis, DMSO, and L-metionin have been reported (121).

Based on these data, in a recent back to nature move, modern man is searching for natural products with medicinal properties, particularly those from plants and bees (26, 81,126).

Several plants produce resinous exudates with strong anti-microbial and anti-necrotic properties (83), in addition to impermeability provided by populus - a substance from Populus sp (16). Bees collect resin exudates from certain plants and add their secretion, wood fragments, pollen, and wax; this product from bees and plants is called propolis. The word propolis comes from the Greek pro meaning in defense of and polis city, representing defense of bee cities (or beehives). Propolis has been used in folk medicine since primeval times (43). It was used in Egyptian rituals to embalm their dead (74); as violin varnish in Italy in the 17th century (56,83); and as a local antiseptic for umbilical cords in the Middle Ages (76). Nowadays, propolis is still used in homemade remedies and cosmetics (16). Two characteristics of propolis are its smell and its various colors from dark green to brown.

Ethanol, the most commonly used solvent for propolis preparations, and other solvents such as ethylic ether, water, methanol, petroleum ether, and chloroform are used for extracting and identifying many propolis compounds (73,74). Moreover, glycerin, propylene glycol and some solutions have been used in propolis preparations by the pharmaceutical and cosmetic industry (125).

Propolis compounds have recently become the subject of investigation in order to determine its therapeutic application (60); flavonoids are the most biologically active (9,16,53).

However, some hypersensitive reactions induced by propolis and its isolated constituents, especially those derived from cinnamic acids have been reported (49,50-52). Oedema and erithema in the face and hands of violin polishers in Cremona, Italy, are related to contact dermatitis with propolis (83). Allergic reactions caused by propolis have also been reported (52).

Propolis has low acute oral toxicity, as shown by the LD50 tested in mice (2.000 to 7.300 mg/kg) and flavonoids evaluated in rats (8.000 to 4.000 mg/kg) (54). No side effects have been seen in oral administration to mice higher than 4.000 mg/kg/day for two weeks (23) and in drinking water at 1.400 mg/kg/day and for 90 days, and to rats at 2.740 mg/kg/day for 60 days (57). On the other hand, intraperitoneal administration of ethanolic propolis extracts has slight effects on animals under narcotic-induced hypothermia. Propolis oral administration does not show any significant alteration in some important enzyme levels in rats (116).

Considering that propolis is a complex mixture, synergistic interactions between its compounds must also be considered as an important factor in its anti-inflammatory activities (18,64,131,132).

In the last 20 years, there has been increased commercial interest (14) in propolis use due to its therapeutic properties (17,71,87). Propolis is commercially found in sprays, ointments, capsules, capillary lotions, and toothpastes (16) because of its bacteriostatic activity and pharmacological properties.

Propolis can be used in decubitus scabs, (2) dermatitis, psoriasis, itching, burning, (17), leg ulcers, (78) simplex and genital herpes (33), and in oral hygienic odontology (71,87). Propolis extracts show: anti-bacterial (27,46,130,132), anti-viral (1,113), anti-fungal (29), anti-protozoan (23,120) antioxidant (95,110,111), and anti-tumor properties (47,77,109). Propolis is also effective in antibody formation in immunized animals (108) as a hepatoprotective (69) and for its hypertensive effects (3). It increases natural killer activity against tumor cells and shows anti-microbial action against GRAM-positive bacteria (114). Propolis minimal inhibitory concentration (MIC) has been performed in order to establish the anti-microbial activity profiles of its extracts (36,115). Nowadays, many in vitro and in vivo experiments are performed with propolis ethanolic extracts (PEE) and propolis aqueous extracts (PAE) to confirm its anti-inflammatory activity. PAE anti-inflammatory effects was observed in platelet aggregation inhibition, in vitro PG biosynthesis, and adjuvant-induced paw oedema in vivo, when orally administered and in a dose-dependent-manner (58). PAE inhibits acute inflammation model (PGE2-induced paw oedema) and chronic inflammation (formaldehyde-induced arthritis). However, PAE has no effect against carrageenin-induced paw oedema and Freunds adjuvant-induced arthritis (29).

The anti-inflammatory effects of orally administered PEE were observed in the inhibition of carrageenin-induced oedema, Freunds adjuvant-induced arthritis, cotton-pellet-induced granuloma, and vascular permeability and analgesia in a dose-dependent manner (94).

Propolis suppresses the generation of LOX and COX during acute peritonitis induced by in vivo zimosan and in vitro murine peritoneal macrophages, inhibiting in vivo high production of LTB4 and LTC4. However, its oral supplements do not affect PGE2 generation in zimosan-treated mice ex vivo or in vitro, increasing LT and PG production by peritoneal macrophages (81). Propolis extracts act on host non-specific immunity activating macrophages, inducing H2O2 release, and inhibiting nitric oxide generation in a dose-dependent manner (90).

CAPE action was observed on the generation of several cell oxidative processes: a) myeloperoxidase activity (MPO) by PMN infiltration in mouse ear induced by tumor promoter; b) respiratory burst of human PMN; and c) formation of oxide-base in epidermal DNA isolated in mice treated in vivo (41). It was also observed that CAPE and CA are strong LOX inhibitors, suppressing leukotriene production by peritoneal macrophages. Their action on LTC4 was smaller in vivo (81). Quercetin inhibits LOX, and at high concentrations blocks COX. Naringenin only inhibited LTC4 causing weakness. Propolis constituents have the ability to produce free radicals in inflammatory events (53) including neutrophil chemiluminescence (63). However, some models of inflammation induction (where phlogogen is attenuated in vitro) do not demonstrate propolis extract effects on already established inflammation (68,119).

All these data have demonstrated the strong and different inhibitory action of several propolis preparations or its isolated constituents on inflammation events. However, its anti-inflammatory effects depend mainly on the administration route and its potency (81).

In an attempt to establish quality standards for propolis containing products, physical-chemical analysis studies have not been sufficient (72,131,132,133) mainly for the great variety of compounds detected in propolis from tropical regions (75). These standards should depend specifically on their different pharmacological activities (79).

There are few studies reporting on in vivo anti-inflammatory activity of propolis containing products (80). Based on these data, an evaluation of the anti-inflammatory potential of commercial propolis containing products from several phyto-geographic origins is of major importance for its indication in inflammatory processes.

ACKNOWLEDGEMENTS

The authors are grateful to Ms. Lívia Simão de Carvalho, a medical student at Federal University of Mato Grosso do Sul, Campus of Dourados.